A key player in mast cell activation is the high affinity IgE receptor, FceRI. While much is known about the chain of events initiated by crosslinking of FceRI through multivalent antigen, there is a fundamental gap in our understanding of how protein dynamics facilitate signaling. Our long term goal is to understand how the dynamic and stochastic behavior of protein-protein interactions influences signal propagation. The objective of this proposal is to directly quantify protein interactions and changes in the surrounding membrane environment during early events in FceRI signaling. Our central hypothesis is that the spatiotemporal coordination of FceRI signaling complex formation modulates the strength and duration of the cellular response. The rationale for the proposed research is that understanding the role of protein-protein interaction dynamics in signaling is the next step in understanding how the cell shapes the strength and quality of an immune response. The goal of this proposal is to develop novel FRET measurements based on fluorogen activating peptide (FAP) technology that will provide improved signal detection for measuring protein-protein and protein-lipid interactions in living cells. This new methodology will complement our ongoing projects by allowing us to address additional questions about FceRI signaling dynamics that are simply unaddressable with conventional methods. The approach is innovative because we will develop methodology for imaging biochemical events at the molecular level that will allow us to obtain dynamic information about signaling events that cannot be determined using traditional biochemistry techniques. The proposed research is significant because the quantitative information that we propose to obtain has not been directly measured before and will bring new perspectives to the cell biology community. Furthermore the FAP-based FRET methods will be broadly applicable to other cell signaling pathways. The information we gain about FceRI signaling will help to fill the gaps in our knowledge of how FceRI initiates signaling. Ultimately, we expect that this information will open new avenues for drug design that target protein interactions and localization.